In spite of the major advances that have occurred in recent years, the normal mechanisms in the intracellular regulation of smooth muscle is incompletely understood The. long-term objectives of this project are to obtain a complete understanding of the mechanism(s) responsible for contraction of smooth muscle and to apply this knowledge to understand the changes that occur in pathophysiological states affecting smooth muscle function. This project is based on the hypothesis that two Ca2+ dependent regulatory systems act in parallel, one for the rapid development of stress by rapidly cycling, phosphorylated crossbridges, and the other for the slow development and maintenance of stress by slowly cycling, unphosphorylated latchbridges. The specific aims of this application are to: 1) determine if an increase in intracellular free calcium (Ca2+) activates smooth muscle contraction by two independent pathways; 2) determine the protein system(s) involved in these Ca2+ dependent pathways; 3) determine if the source of the activator Ca2+ influences the specific regulatory mechanism that will support contraction; and 4) determine if these two putative Ca2+ regulatory mechanisms are present in non-vascular smooth muscles in a quantitatively similar ratio. Actomyosin from swine aorta, and "detergent skinned" and intact fibers of swine carotid artery will be the primary models for this investigation. Experiments will be performed with novel specific inhibitors of the MLC kinase or with the ATP analog, CTP. Measurements will include Ca-+ and Mg2+ activation of ATPase and superprecipitation of actomyosin, and stress, ML phosphorylation, crossbridge cycling rate and attachment in intact and detegent skinned fibers. The proteins involved in this second Ca2+ dependent system will be identified by binding of radiolabelled compounds (Ca, Mg, W-7, and calmodulin) to proteins on nitrocellulose membranes and then separated by column chromatography. The sources of activator Ca2+ will be isolated pharmacologically and the effects of various Ca2+ sources on stres, MLC phosphorylation and crossbridge cycling rate examined. The results of these studies will be compared to studies using bovine trachealis and rat intestinal smooth muscle to determine the Ca2+ dependent regulation of other smooth muscles. The information gained from this application will provide direct evidence for the presence of two Ca2+ dependent regulatory systems in smooth muscle, localize and characterize the protein(s) involved in this regulation, and discern the relationship between the source of activator Ca2+ and the mechanism by which smooth muscle contracts. Knowledge concerning the regulation of smooth muscle contraction is a necessary prerequisite for understanding the role of alterations in smooth muscle associated with pathophysiological disease states.